Chemotaxis occurs during a number of key physiological events, including angiogenesis, embryonic development and wound healing. It also contributes to disease progression in pathological conditions such as cancer metastasis and arthritis. The goal of the current proposal is to reveal how biochemical reactions and physical phenomena such as membrane deformation interact with one another in regulating chemotaxis. Specifically, we will focus on elucidating the role of a superfamily of membrane deforming proteins, Bin/Amphiphysin/Rvs (BAR), in distinct steps of chemotaxis. These steps include sensation of an extracellular chemical gradient, cellular amplification of the input stimulus, polarization of intracellular signaling events, and actuation of cell motility. For three BAR proteins that we already shown are involved in cell migration via gain- and loss-of-function studies, we will precisely determine how each of these BAR proteins is required for chemotaxis by performing biochemical and cell biological assays along with computational modeling. In particular, we execute the loss-of-function studies of the three BAR proteins to determine their role in any one of the aforementioned steps of chemotaxis, with an emphasis on the polarization process by performing chemotaxis and chemokinesis assays (Aim 1). We will then reveal the role of these BAR proteins specifically in one of the core polarization programs, namely a positive feedback loop that is known to consist of several signaling molecules (Aim 2). This will be achieved by conducting chemotaxis assays using both shallow and steep chemical gradient, as well as an imaging-based assay we developed to quantitatively measure the extent of feedback actuation. We also investigate sufficiency of BAR-induced membrane deformation in the positive feedback using newly established tools that can deform membrane inside living cells within seconds. Collectively, Aims 1 and 2 will characterize the crosstalk between biochemical and physical factors during the positive feedback process that drives cell polarization. We will then reveal how BAR proteins mediate the cooperative actuation of the positive feedback loop at a molecular level (Aim 3). Based both on previous reports and our own recent findings, we hypothesize that signaling molecules such as PI3K can sense membrane curvature, and therefore accumulates at local sites on the plasma membrane which have been bent by BAR proteins. To test this hypothesis, we will perform two experiments: an in vitro liposome binding assay and a cell-based localization assay. To further elucidate this non-intuitive, cooperative process on a quantitative level, parameters derived from these wet experiments will be integrated into a computational model. Combined, the work outlined here represent powerful means by which we can explore crucial, but often understudied, aspects of chemotaxis. More specifically, it will reveal the central role that membrane-deforming proteins play during cell polarization, and offer molecular insights into pathophysiological conditions where dysfunction of chemotaxis plays a significant role in disease progression, such as cancer metastasis and arthritis.